Project Summary Infants born to HIV-infected mothers are at high risk for HIV acquisition via breastfeeding. Yet, the WHO recommends exclusive breastfeeding for HIV-exposed infants since replacement feeding is associated with high morbidity and mortality. Although antiretroviral therapy can reduce transmission, the risk is high compared to that of heterosexual sex. Therefore, HIV-exposed, breastfed infants are an important target group for an HIV vaccine. HIV-exposed infants display altered vaccine responses compared to unexposed infants. Animal models have demonstrated that the development of certain T cell subsets, both in the mucosa and systemically, is determined by the presence of specific microbes in the gut, and may be important in determining adaptive immunity. In humans, there are many reasons to postulate that the gut microbiome of HIV-exposed uninfected (HEU) infants differs from that of HIV-unexposed (HU) infants, since their mothers have altered gut microbiome, and they are exposed to antibiotics and antiretrovirals during pregnancy and postpartum. This proposal tests the hypothesis that in HEU infants who are breastfed, the gut microbiome differs in diversity, structure and function, from that of HU infants, and that specific gastrointestinal microbial profiles correlate with immune response to early childhood vaccines. This proposal will utilize an already funded, ongoing cohort designed to identify correlates of immune protection associated with exclusive breastfeeding, the InFANT cohort, that is enrolling 500 HIV-infected mothers and their breastfed infants, and 150 HIV-negative mother- infant pair controls. As part of the InFANT study, comprehensive sample and data collection is occurring with detailed immunological assessments including vaccine responses. This proposal will utilize the existing infrastructure and samples to characterize the infant gut microbiome of 150 HEU versus 75 HU infants via 16S rRNA sequencing, and its succession in the first 9 months of life in breastfed infants, i.e. in the first week of life (prior to initiation of cotrimoxazole prophylaxis), 7, 15 and 36 weeks of age (Aim 1). The study will relate the microbial composition and its corresponding function (Aim 2) using shotgun metagenomics, proteomics, and metabolomics, to T cellular responses to BCG vaccination in 150 HIV-exposed infants. Finally, will translate associations between gut microbes and their byproducts will be tested for causative roles in vaccine immunity by colonizing germ free mice with specific organisms, proteins and metabolites from our preliminary data and Aim 2, and then assessing their consequent response to BCG using state-of-the-art tetramer technology (Aim 3). Aim 3 will also delve into mechanisms of microbiome-vaccine immunogenicity interactions by assessing global B and T cell subsets in the mesenteric lymph nodes and periphery. This study brings together experts in clinical, pre-clinical, immunologic, mass-spectrometric and microbiome analyses. This joint approach will be used to investigate in detail the mechanisms through which gut microbiome alters immune responses of HEU infants, leading to improved development of HIV vaccines for these vulnerable neonates.